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CN-121991906-A - Efficient expression and crystallization method of Chlamydomonas reinhardtii HydA2 protein

CN121991906ACN 121991906 ACN121991906 ACN 121991906ACN-121991906-A

Abstract

The invention provides a high-efficiency expression method of Chlamydomonas reinhardtii HydA2 protein, which comprises the following steps of constructing a pET-32a-TEV-HydA2 recombinant expression vector, obtaining a positive recombinant strain, inducing expression of the Chlamydomonas reinhardtii HydA2 protein, and purifying the Chlamydomonas reinhardtii HydA2 protein. The invention also provides a crystallization method of the Chlamydomonas reinhardtii HydA2 protein, which takes the Chlamydomonas reinhardtii HydA2 protein prepared by the method as a raw material. According to the invention, a pET-32a vector is modified through homologous recombination, a pET-32a-TEV expression system is constructed, and escherichia coli expression, induction and purification are combined, so that efficient soluble expression and activity retention of the Chlamydomonas reinhardtii HydA2 protein are realized, the problems of easy folding error, activity loss and low expression level of the Chlamydomonas reinhardtii are solved, an effective crystallization system is established, and a foundation is laid for structural analysis and research on hydrogen production of green algae.

Inventors

  • NIU MENGYING
  • SUN YU
  • LIU LIN

Assignees

  • 安徽大学

Dates

Publication Date
20260508
Application Date
20260211

Claims (10)

  1. 1. The efficient expression method of the Chlamydomonas reinhardtii HydA2 protein is characterized by comprising the following steps: (1) Construction of pET-32a-TEV-HydA2 recombinant expression vector Preparing a target gene fragment with BamH I/Sal I double enzyme cutting sites by taking a Chlamydomonas reinhardtii HydA2 gene with a nucleotide sequence shown as SEQ ID NO.1 as a template, connecting the gene fragment with a pET-32a vector after BamH I/Sal I double enzyme cutting to obtain a pET-32a-HydA2 recombinant plasmid, adding a TEV enzyme cutting site at the downstream of the BamH I enzyme cutting site of the recombinant plasmid by a homologous recombination technology, and obtaining the pET-32a-TEV-HydA2 recombinant expression vector after identification and sequencing; (2) Acquisition of Positive recombinant Strain Transforming the pET-32a-TEV-HydA2 recombinant expression vector into an escherichia coli competent cell, culturing, and then picking a single colony for identification to obtain a positive recombinant strain; (3) Induction expression of Chlamydomonas reinhardtii HydA2 protein Performing amplification culture on the positive recombinant strain, adding IPTG for induction culture, and centrifugally collecting thalli after the culture is completed; (4) Purification of Chlamydomonas reinhardtii HydA2 protein And (3) subjecting the collected thalli to cracking, crushing and centrifugation, collecting supernatant, and sequentially carrying out fine purification by Ni-NTA affinity chromatography and a chromatography system to obtain the target desired Chlamydomonas reinhardtii HydA2 protein.
  2. 2. The expression method of claim 1, wherein in the step (1), the preparation method of the target gene fragment is that artificially synthesized Chlamydomonas reinhardtii HydA2 gene is used as a template, and primers F1 and R1 are used for PCR amplification, wherein the nucleotide sequences of the primers F1 and R1 are respectively shown as SEQ ID NO.2 and SEQ ID NO. 3.
  3. 3. The method according to claim 1, wherein in the step (1), the nucleotide sequence of the TEV cleavage site is shown as SEQ ID NO.4, the double-stranded fragment formed by the reverse complementary primers F2 and R2 is used as a target fragment by the homologous recombination technology, the target fragment is subjected to recombination reaction with the pET-32a-HydA2 recombinant plasmid which is subjected to single cleavage linearization by BamH I, and the nucleotide sequences of the primers F2 and R2 are shown as SEQ ID NO.5 and SEQ ID NO.6 respectively.
  4. 4. The method according to claim 1, wherein in the step (2), the E.coli competent cells are DH 5. Alpha. Competent cells, and the step (2) is characterized by at least one of PCR identification and double digestion identification.
  5. 5. The method according to claim 1, wherein in the step (3), IPTG is added when the positive recombinant strain is grown to a bacterial liquid OD 600 of 0.6-0.8, the final concentration of IPTG is 0.4mM, and the culture is induced at a low temperature of 16 ℃ for 12-16 hours.
  6. 6. The method according to claim 1, wherein in the step (4), the lysis buffer used for the lysis is a mixture of 20mM Tris-HCl and 200mM NaCl, pH 7.5, the disruption is ice-top ultrasonication, and the chromatography system is Superdex 200 chromatography.
  7. 7. The method according to claim 1, wherein in the step (4), ni-NTA affinity chromatography is performed by imidazole gradient elution, and the elution buffer is a mixed solution containing 20mM Tris-HCl, 150mM NaCl and 20-500 mM imidazole.
  8. 8. A crystallization method of chlamydomonas reinhardtii HydA2 protein, which is characterized in that chlamydomonas reinhardtii HydA2 protein prepared by the method of any one of claims 1 to 7 is used as a raw material, and crystal screening and optimization are performed by a sitting-drop method to obtain chlamydomonas reinhardtii HydA2 protein crystals.
  9. 9. The crystallization method according to claim 8, wherein the Chlamydomonas reinhardtii HydA2 protein is concentrated to 6mg/mL, and subjected to centrifugal filtration treatment, and then subjected to crystal screening, wherein the sitting-drop method adopts a 48-well crystal plate, 200 μl of cell solution is added into a tank, a protein sample in the small hole and the cell solution are mixed according to a volume ratio of 1:1, and the cell solution is cultured and observed after sealing.
  10. 10. The crystallization method according to claim 8, wherein the crystallization growth condition of the crystals is that 0.2M lithium sulfate, 0.1M BIS-TRIS, 25% (w/v) polyethylene glycol 3350 are used as a pool solution, chi Ye pH 6.5, and the crystals are cultured at 18 ℃ for 7 days.

Description

Efficient expression and crystallization method of Chlamydomonas reinhardtii HydA2 protein Technical Field The invention relates to the technical field of genetic engineering and protein engineering, in particular to a method for efficiently expressing and crystallizing HydA2 protein of Chlamydomonas reinhardtii. Background The green algae can extract protons and electrons from water through a sunlight-driven biological process, and chemical energy is converted into green and environment-friendly hydrogen (H 2), and the core of the process is [ FeFe ] -hydrogenase expressed by the green algae, which is used as a high-activity biocatalyst, can catalyze the reversible reduction of protons into molecular hydrogen, and is connected with a photosynthetic electron transfer chain through ferredoxin PETF. The [ FeFe ] -hydrogenase has the dual functions of oxidizing hydrogen to supply energy to the organism and capturing and releasing electrons in the organism, and the biotechnological application of preparing H 2 from renewable resources becomes a potential sustainable solution for coping with climate change due to high catalytic efficiency. To date, there are many undefined mechanisms of algae hydrogen metabolism, and most of the characterized chlamydomonas hydrogenases consist of only the active site domain (H cluster), no additional domain or iron sulfur (FeS) cluster. The active site consists of a "standard" [4Fe4S ] -cluster (4 FeH), linked by bridging cysteines to a unique [2Fe2S ] -cluster (2 FeH), which coordinates several non-proteogenic ligands (two CNs, three CO and one bridging aza-disulfate adt). In addition to the coordination of the so-called H-clusters with the four cysteine ligands, the protein environment around the active site (secondary ligand sphere) influences ligand geometry, redox state and H-cluster stability through various electrostatic contacts and H-bond networks. Chlamydomonas reinhardtii (Chlamydomonas reinhardtii) is a typical model organism of green algae and encodes two [ FeFe ] -hydrogenases (HydA 1, hydA 2) capable of independently catalyzing proton reduction to H 2, wherein HydA1 is a main functional protein generated by algae H 2, and is connected with a photosynthetic electron transfer pathway through a plant type ferredoxin PETF, while HydA2 bears about 25% of hydrogen production function in algae metabolism, and a specific action mechanism is not clear. Chlamydomonas reinhardtii HydA2 is taken as biferrohydrogenase, the catalytic activity of the HydA2 is superior to that of [ NiFe ] -hydrogenase, and the HydA is an important research object in the field of hydrogen production catalysts, but the Chlamydomonas reinhardtii hydrogenase has extreme sensitivity to oxygen, and the characteristic results in extremely low hydrogen production efficiency, so that the HydA becomes a core bottleneck for restricting the practical application of the Chlamydomonas reinhardtii hydrogen production technology. Green algae possess multiple ferredoxin subtypes, can regulate electron flow in different environments through specific interactions with different metabolic redox partners, and differ in the type of ferredoxin bound by different hydrogenases. At present, the preparation of the efficiently expressed Chlamydomonas reinhardtii HydA2 avoids the inhibition of the hydrogenase activity by regulating the oxygen concentration, is a key research direction for improving the photosynthetic hydrogen production efficiency of green algae, and meanwhile, the space structure of the Chlamydomonas reinhardtii HydA2 is not resolved, but the protein structure is resolved, so that the support can be provided for the research of the physiological and biochemical pathway of the Chlamydomonas reinhardtii HydA2, a method for improving the oxygen tolerance of the Chlamydomonas reinhardtii HydA is further found by assistance, and the improvement of the hydrogen production efficiency is finally realized. Therefore, the efficient active expression of the Chlamydomonas reinhardtii HydA2 protein is realized, the exploration of crystallization conditions is completed, and the method is an important research direction for improving the photosynthetic hydrogen production efficiency of the green alga. PET-32a is a commonly used prokaryotic expression vector, and a self-carried thioredoxin label (Trx) can assist eukaryotic proteins to be correctly folded in escherichia coli to form a natural conformation, has the functions of resisting oxidization and regulating disulfide bond reduction, can protect oxygen-sensitive proteins from oxidative damage, meets the heterologous expression requirement of HydA2 proteins, can realize the accurate assembly of DNA fragments by a homologous recombination technology, and provides technical support for the directional transformation of the vector. Based on the method, the pET-32a vector is modified by utilizing the homologous recombination technology, so that the efficient expression and